Functional analysis of acetylcholine receptor using muscarinic receptor knockout mice.

使用松井博士（千叶科学研究所）开发和提供的毒蕈碱乙酰胆碱受体（mAChR）敲除（KO）小鼠。免疫应答调节：免疫后第14天，M_1/M5-KO小鼠血清抗原特异性IgG_1水平显著低于野生型小鼠。此外，豆豆素a可诱导野生型小鼠乙酰胆碱合成酶ChAT、白细胞介素-2和c-fos mRNA的表达。与野生型小鼠相比，M_3 ko小鼠的这些基因表达显著降低。这些结果提示M_1/M_5和M_3 mAChR可能参与了淋巴细胞功能的调节。放松膀胱肌肉：在野生型小鼠中，甲乙醇（CCh）以剂量依赖的方式放松去甲肾上腺素诱导的收缩。然而，在M_3-KO小鼠中，CCh的松弛作用减弱，而在M_2-KO小鼠中则没有变化。此外，吲哚美辛可拮抗cch诱导的反应。此外，PGE_2诱导了更多的d浓度依赖性弛豫。这些结果提示M_3 mAChR和PGE_2参与了膀胱肌肉的松弛。mAChR配体性质的变化：对M_2-KO小鼠口服奥氧布宁（Oxy）和索利那新（Sol）后，对膀胱和颌下腺的mAChR结合活性进行了表征。在M_2-KO小鼠的膀胱和颌下腺中，Oxy和Sol的体外mAChR结合活性差异不大，表明两者组织中残留的（主要是M_3） mAChR具有相同的亲和力。与口服Oxy后相比，口服Sol对M_2-KO小鼠膀胱mAChR的结合活性明显增强，而对外分泌腺mAChR的结合活性明显减弱。这些数据表明，在体内条件下，口服Sol与膀胱M_3 mAChR的结合可能比外分泌腺更大。口服固液对膀胱有较高的选择性，可能有利于治疗膀胱过动症。少

Muscarinic acetylcholine receptor (mAChR) knockout (KO) mice, which were developed and supplied by Dr Matsui (Chiba Institute of Science), were used. Regulation of Immunological response: On the 14th day after immunization, serum antigen specific IgG_1 levels in M_1/M5-KO mice were significantly lower than those of wild-type mice. In addition, concanavalin A-induced ChAT, an ACh-synthesizing enzyme, interleukin-2 and c-fos mRNA expression in wild-type mice. These gene expressions were significantly reduced, in M_3 KO-mice, when compared with those of wild-type mice. These findings suggested that M_1/M_5 and M_3 mAChR may involved in regulation of lymphocyte function. Relaxation of Urinary bladder muscle: In wild-type mice, carbachol (CCh) relaxed noradrenaline-induced contraction in a dose-dependent manner. However, relaxation by CCh was decreased in M_3-KO mice, but not changed in M_2-KO mice. In addition, CCh-induced response was antagonized by indomethacin. In addition, PGE_2 induce … More d concentration dependent relaxation. These findings suggest that M_3 mAChR and PGE_2 are involved in relaxation of urinary bladder muscle. Changes in mAChR ligand properties: Characterization of mAChR binding activity in the bladder and submandibular gland after oral administration of oxybutynin (Oxy) and solifenacin (Sol) to M_2-KO mice was performed. There was little difference between bladder and submandibular gland of M_2-KO mice in in vitro mAChR binding activities of Oxy and Sol, suggesting equal affinity to the residual (predominantly M_3) mAChR in both tissues. In contrast, compared with that after oral Oxy, oral Sol exerted significantly greater binding activity to the bladder mAChR of M_2-KO mice, whereas oral Sol exhibited significantly smaller binding activity to the exocrine gland mAChR. These data indicate that oral Sol, unlikely oral, may exert greater binding to M_3 mAChR of bladder than exocrine gland under in vivo condition. Oral Sol may be advantageous for the treatment of overactive bladder, in terms of high selectivity to bladder. Less